JPH0641933B2 - Two-dimensional electrophoresis - Google Patents

Description

TECHNICAL FIELD The present invention relates to a two-dimensional electrophoresis method, in particular, a two-dimensional electrophoresis method for a protein sample using a polyacrylamide gel and an apparatus for carrying out the method. .

Conventional technology Conventionally, a two-dimensional electrophoresis method in which a sample is electrophoresed in a one-dimensional gel, the resulting one-dimensional sample gel column is placed on the upper end of the two-dimensional gel, and further electrophoresed. In, for example, when polyacrylamide gel is used as the one-dimensional and two-dimensional gels and Escherichia coli ribosomal proteins are separated and detected, the sample protein is added as a solution to the cylindrical one-dimensional gel and the components are separated by the electrophoresis (one-dimensional separation). Then, the columnar gel fixed in that state was placed on the upper end of the different-dimension gel to perform two-dimensional migration.

Problems to be Solved by the Invention In this case, there is a drawback in that free radicals remaining in each gel before electrophoresis chemically change the sample protein during electrophoresis and consume a part of the sample. Further, in each gel during the electrophoretic treatment, there was also a drawback that the protein molecules in the sample were SS-crosslinked due to the oxidation of cysteine residues, dimerized and became difficult to be separated. Further, as a problem peculiar to the two-dimensional gel, the basic low molecular weight protein is
There was an inconvenience that the concentration process was prolonged due to overlapping with the migration front and the separation was insufficient.

Further, since the columnar type one-dimensional separating agent gel is placed on the upper end of the two-dimensional gel, a gap is formed on both sides of the contact surface of both gels, which has been a cause of disturbance of migration.

Means and Actions for Solving Problems In order to solve these problems, the present invention allows a sample component to be electrophoresed in a one-dimensional gel and the resulting one-dimensional sample gel to be the upper end of the two-dimensional gel. In a two-dimensional electrophoresis method, which consists of placing the sample on a plate and further electrophoresing it, the sample is concentrated in a pretreatment rod-shaped gel by electrophoresis to form a zero-dimensional sample gel. A two-dimensional gel characterized by preparing a one-dimensional separating agent gel to be electrophoresed by inserting it at a desired level position, which is notched, in order to carry out one-dimensional electrophoresis, thereby placing it on the upper end of the two-dimensional gel for further electrophoresis. This is an electrophoretic method.

According to the present invention, in the above-mentioned constitution, so-called zero-dimensional electrophoresis is performed as a pre-stage of the one-dimensional electrophoresis, so that the sample is concentrated so as to avoid the protein molecules in the sample from individually encountering free radicals. This enables effective one-dimensional migration and further two-dimensional migration.

The present invention also allows the pretreatment rod-shaped gel and the one-dimensional gel to have a rectangular cross-section, so that the sample gel and the one-dimensional separating agent gel adhere to the one-dimensional gel and the two-dimensional gel without much space. It is something that is done. The adhesiveness of such gel connection completely eliminates the drawback of the conventional one-dimensional gel preparation method in a cylindrical tube in which gaps are formed on both sides of the connection surface and migration is disturbed.

The present invention, further, in analyzing the protein sample using the rod-shaped gel, which is made of polyacrylamide, one-dimensional gel and two-dimensional gel, before the addition of the corresponding sample solution or gel in each gel, before migration, By migrating a radical scavenger composed of a charged body for removing free radicals remaining in each gel, the sample is prevented from being consumed by free radicals during the migration on each gel.

The present invention further analyzes the protein sample by using the rod-shaped gel, the one-dimensional gel and the two-dimensional gel, each of which is made of polyacrylamide. In each gel, a corresponding sample solution or gel is added to cause electrophoresis and strong reduction. By running a reducing agent composed of a charged body for maintaining the target state, protein molecules in each gel are prevented from SS-crosslinking and dimerization.

In the present invention, when analyzing a protein sample using the rod-shaped gel, one-dimensional gel and two-dimensional gel, both of which are made of polyacrylamide, the basic radical accelerator added to the two-dimensional gel is removed by pre-migration. At the same time, a strong acid solution is added to the negative electrode buffer to increase the acidity of the gel, thereby improving the separation ability for basic low molecular weight small proteins.

Description of Examples Zero-dimensional migration Zero-dimensional migration as referred to here is a process of concentrating and forming a sample gel inserted into a one-dimensional gel by electrophoresis instead of injecting a sample solution into the one-dimensional gel. That is. In this zero-dimensional electrophoresis, a gel container GC ₀ having one or more rows of prismatic gel chambers (1) as shown in FIG. 1 (a) is formed by an upper buffer tank (2) and a lower buffer tank (3). ), The gel chamber (1) contains, for example, a polyacrylamide gel as an electrophoretic medium for a protein sample, and the sample solution is introduced from above.
In this case, an appropriate voltage is applied between the anode A provided in the upper buffer tank (2) and the cathode B provided in the lower buffer tank (3). Before this migration, 2-mercaptoethylamine as a radical scavenger is pre-migrated on the polyacrylamide gel, whereby the consumption of the sample protein can be prevented. Further, during migration of the sample solution, cysteine as a reducing agent is also co-migrated, thereby preventing SS cross-linking of the protein molecule. The sample thus electrophoretically concentrated is taken out from the gel container GC ₀ or taken out by disassembling the container, and then the appropriate portion of the concentrated gel portion (4) is cut out to prepare a sample gel piece SG for one-dimensional gel. And

The upper buffer tank used for this zero-dimensional migration
(2) and the lower buffer tank (3) have the same structure as the upper buffer tank and the lower buffer tank for one-dimensional electrophoresis described below, respectively. The only difference in the mechanism is that a gel container having a shorter total length than the above is used. The distance between both plates of the gel container GC ₀ , that is, the thickness of the gel is about 3 mm in this case, but it can be made from about 1 mm to more than 3 mm according to its use. The same applies to the gel thickness for one-dimensional migration and two-dimensional migration.

One-dimensional migration Gel container GC ₁ for one-dimensional migration is a prismatic column as shown in Fig. 2 (a), that is, a plurality of rows of gel chambers having a rectangular cross section.
In addition to having (10), a sample insertion window (11) communicating with each gel chamber (10) is formed at a relatively high level position on one side surface. The sample gel piece by the zero-dimensional treatment has a height slightly lower than the vertical dimension of this window, but since the zero-dimensional and this one-dimensional gel chamber width and depth are the same, the gel chamber from this window (10) Each of the sample gel pieces SG inserted in the section enters the excision portion corresponding to this window in the electrophoretic polyacrylamide gel in the same room, and adheres well to the entire upper and lower cut ends. SGC shown in FIG. 2 (b) is a sample gel cover plate, wherein each window of the gel container GC ₁
It has a plurality of window stoppers (12) corresponding to (11). Each window plug (12) is inserted into each corresponding window (11), thereby supporting and covering the side surface of the sample gel piece SG at the corresponding position in the gel chamber (10). The gel container GC ₁ equipped with the sample gel cover SGC is the third
As shown in the figure, the upper and lower ends are attached to the upper buffer tank ABV ₁ and the lower buffer tank CBV ₁ , and an appropriate voltage is applied between the anode A and the cathode C to perform the electrophoretic separation of the sample gel. Is. The sample gel cover SGC is attached to the gel container GC ₁ by using a clip (13) as shown in FIG. 4, for example. Also, the upper buffer tank ABV ₁ and the lower buffer tank C
The internal structure of BV ₁ is also as shown in FIG.

The polyacrylamide gel in the above one-dimensional migration is
Before inserting the sample gel piece SG, 2-mercaptopropionic acid as a radical scavenger is pre-migrated, and further, during migration of the sample gel, the same 2-mercaptopropionic acid as a radical scavenger is used as a reducing agent. Are simultaneously electrophoresed to maintain a strong reducing state in the gel.

As described above, the method of preparing the sample gel piece separately from the one-dimensional gel and inserting it into the one-dimensional gel is a unique method adopted only in the present invention, whereby the pre-electrophoresis treatment of the radical scavenger is performed. Together with this, the problem of sample consumption due to residual radicals has been solved.

Two-dimensional migration A two-dimensional migration gel container is performed by combining three types of gel container plates LP, MP, and RP as shown in FIG. 5, and connecting the gel container structure thus formed to the upper and lower buffer tanks. . When two or more two-dimensional gel chambers are used, the space between the MP-MP plate surfaces and the left container plate LP
Is formed between the right side surface of the container plate MP and the left side surface of the corresponding container plate MP, and between the left side surface of the right side container plate RP and the right side surface of the corresponding container plate MP.

The cross section of the structure in which the container structure GC ₂ is combined with the upper buffer tank ABV ₂ and the lower buffer tank CBV ₂ is as shown in FIG. 6, and each gel chamber has an allowance for mounting the one-dimensional migrated gel on the upper end. The gel BG for two-dimensional electrophoresis is inserted while leaving the above. The slot D ₁ is formed at the upper end of the container plate MP, and the upper end outer surfaces of the left container plate LP and the right container plate RP and the upper buffer tank AB are formed.
A similar slot D ₂ is formed between the protrusion formed at the lower end of V ₂ . The one-dimensional migrated gel SG ₂ is inserted from the upper end of each gel chamber of the container structure GC ₂ , and
Gauze is applied to the upper end of the container structure GC ₂ , and the flat wedge PW is inserted into the slots D ₁ and D ₂ from above the gauze G, and the gauze tensioned by this inserts the one-dimensional migrated gel SG _{2 into} the gel. The upper edge is pressed so that it is in close contact with the upper end of the two-dimensional electrophoresis gel BG.

FIG. 7 shows the internal structure of the fastening member H for maintaining the container structure GC ₂ , the upper buffer tank ABV ₂ and the lower buffer tank CBV ₂ .

Also in this two-dimensional migration, residual free radicals are removed by migrating 2-mercaptoethylamine as a radical scavenger on the polyacrylamide gel, which is the migration medium, before migration of the sample gel column SG ₂ component, and During migration of the column component of the sample gel, cysteine was co-migrated together as a reducing agent having a charge to maintain a strong reducing state in the two-dimensional gel. at the same time,
By removing the basic radical promoter in the gel by pre-electrophoresis and adding hydrochloric acid to the lower part, that is, the negative electrode buffer tank CBV ₂ , the pH of the gel was lowered from normal 4.5 to about 0.9. To be 3.6.

FIG. 8 is a graph showing the relationship between the molecular weight of sample proteins obtained by carrying out the method of the present invention and the migration distance. In this case, if the difference in the effect of charge on the mobility is eliminated, the logarithmic value of the molecular weight is almost completely proportional to the mobility, indicating that the molecular weight can be measured.

As is clear from the separation model of the two-dimensional gel shown in the graph of FIG. 8 and FIG. 9, from the basic region of the two-dimensional gel, four spots (A, B, C, D) were detected. In FIG. 9, spot A is found at the lower right of L33, spot B is at the lower left of L34, spot C is midway between L29 and L27, and spot D is found just below L32. In addition, A,
B and C are present in the 50S subparticle and D is present in the 30S subparticle. When the method of the present invention was carried out without using a reducing agent, spots A, B and C disappeared, but spots B'and C'were found in the vicinity thereof as B and C disappeared. It was
On the other hand, D forms a spot at the same position regardless of the presence or absence of a reducing agent.

Moreover, when the molecular weights of A, B, C, and D were calculated using the two-dimensional mobility, they were 6400, 4900, 8200, and 5900, respectively. The number of copies was measured by ¹⁴ C amino acid labeling, and the values of 0.6, 0.4, 0.3 and 0.1 were obtained, respectively.

EFFECTS OF THE INVENTION Since the method of the present invention is configured as described above, the free radicals remaining in each electrophoretic gel are effectively removed, and the free radicals are collected based on the concentration of the sample by the so-called zero-dimensional electrophoresis method. To prevent the effects (exhaustion of the sample) of the sample and to maintain the gel in a reduced state, the sample protein splits into two spots, a reduced type and an oxidized type, as in the conventional method, and S-S crosslinking. It prevents the dimerization caused by the above and improves the quantification and resolution. Further, by lowering the pH in the two-dimensional gel from the normal state, the basic low molecular weight protein having high mobility overlaps the migration front and is difficult to be separated. As a result, in the present invention, the logarithmic value of the molecular weight of the sample protein and the mobility show a satisfactory proportional relationship.

[Brief description of drawings]

1 (a) and 1 (b) are vertical cross-sectional views showing the gel container for zero-dimensional electrophoresis of the present invention and its usage, and FIGS. 2 (a) and (b) are gel containers for one-dimensional electrophoresis and FIG. 3 is a perspective view showing a sample gel cover, FIG. 3 is a longitudinal sectional view showing a gel container for one-dimensional electrophoresis and upper and lower electrode tanks attached thereto, FIG. 4 is an exploded perspective view thereof, and FIG. 5 is for two-dimensional electrophoresis. Fig. 6 is a perspective view showing various gel container plates separated from each other. Fig. 6 is a longitudinal sectional view showing a structure in which the gel container plates of Fig. 5 are combined to form a container structure, and upper and lower electrode tanks are attached to it. FIG. 8 is an exploded perspective view thereof, FIG. 8 is a graph showing molecular weight migration distance characteristics obtained by the method of the present invention, and FIG. 9 is a view showing a separation model of a two-dimensional gel. (1) …… Gel chamber (2) …… Upper (anode) buffer tank (3) …… Lower (cathode) buffer tank (4) …… Running gel A …… Anode B …… Cathode SG …… Sample gel SGC …… Sample gel cover ABV …… Upper (anode) buffer tank CBV …… Lower (cathode) buffer tank LP …… Left container plate MP …… Center container plate RP …… Right container plate D …… Slot between containers G …… Gauze PW …… Flat wedge GS …… Gel spacer H …… Fastener

Claims (7)

[Claims] 1. A sample component is electrophoresed in a one-dimensional gel,
In the two-dimensional electrophoresis method, in which the one-dimensional sample gel thus obtained is placed on the upper end of the two-dimensional gel and further electrophoresed, the sample is concentrated in the pretreatment rod-shaped gel by electrophoresis and concentrated to zero. A one-dimensional sample gel is prepared, and the sample gel is inserted at a desired level position cut out in the one-dimensional gel, and one-dimensional electrophoresis is performed, whereby the sample gel is placed on the upper end of the two-dimensional gel for further electrophoresis. A two-dimensional electrophoresis method, which comprises preparing a one-dimensional separated gel. 2. The pretreatment rod-shaped gel and the one-dimensional gel have a square cross section, whereby the sample gel and the one-dimensional separated gel are separated from the one-dimensional gel and the two-dimensional gel, respectively, without leaving a gap. The method according to claim (1), characterized in that they are brought into close contact with each other. 3. When analyzing a protein sample using the rod-shaped gel, the one-dimensional gel and the two-dimensional gel, each of which is made of polyacrylamide, before the electrophoresis is carried out by adding a corresponding sample solution or gel in each gel, Claims characterized in that the radical scavenger consisting of a charged body for migrating free radicals remaining in each gel is migrated to prevent the sample from being consumed by free radicals during migration on each gel. The method according to item (1) or (2). 4. The radical scavenger for the one-dimensional gel contains 2-mercaptopropionic acid, and the radical scavenger for the rod-shaped gel and the two-dimensional gel contains 2-mercaptoethylamine. The method described in paragraph (3). 5. When analyzing a protein sample using the rod-shaped gel, the one-dimensional gel and the two-dimensional gel, each of which is made of polyacrylamide, a corresponding sample solution or gel is added to each gel to cause electrophoresis and strong reduction. The migration of a reducing agent composed of a charged body for maintaining the desired state prevents protein molecules from undergoing SS cross-linking and dimerization in each gel. (1)
~ The method according to any one of (4). 6. The reducing agent for the one-dimensional gel contains 2-mercaptopropionic acid, and the reducing agent for the rod-shaped gel and the two-dimensional gel contains cysteine, Claim (5). The method described. 7. When analyzing a protein sample using the rod-shaped gel, one-dimensional gel and two-dimensional gel, each of which is made of polyacrylamide, the basic radical promoter added to the two-dimensional gel is removed by pre-migration. Along with the addition of a strongly acidic solution to the negative electrode buffer to increase the acidity of the gel, improving the separation ability for basic low molecular weight small proteins claims (1)-(6) The method according to any one of paragraphs.